This application is a 371 of PCT/JP97/02705 filed Aug. 4, 1997.
The present invention relates to an enzymatic method for effectively producing an LH-RH derivative, a useful peptide as a drug.
Luteinizing hormone (LH) and follicle-stimulating hormone (FSH) are released from the anterior pituitary under the control of LH-RH (luteinizing hormone releasing hormone) that is produced at the hypothalamus. LH-RH and derivatives thereof have a secretory activity of gonadotropin and are shown that the sequential administration of LH-RH and its derivatives inhibits gonad functions so that they are applied as drugs to prevent and/or treat diseases such as endometriosis, central precocious puberty, infertility, and prostate cancer. LH-RH and its derivatives that have been applied as drugs include buserelin (JP-B-60-9519 official gazette), goserelin (JP-B-61-13480), leuprorelin (JP-B-53-14072), and napharelin (JP-B-63-56238).
As a method for producing the above described LH-RH and derivatives, a chemical synthesis method by the liquid phase synthesis method wherein peptide fragments with partial sequences corresponding to the polypeptides are formed by the liquid phase or the solid phase method, then each fragment is coupled in liquid phase is known (JP-B-56-47175, JP-A-49-117468, JP-B-57-29462, JP-B-57-25540, JP-B-63-17839, JP-B-63-45398, JP-A-48-40770, JP-A-50-88069, JP-A-49-41375, JP-A-49-41376, JP-A-48-99170, JP-B-52-20996, JP-A-49-35381, JP-B-52-8831, JP-B-57-61268, JP-B-53-14072, JP-B-57-26506, JP-B-60-22720, JP-B-61-13480, and JP-B-3-71439).
However, in the liquid phase synthesis method the solubility changes subtly as the number of amino acid residues of a peptide increases, making it difficult to find an appropriate solvent. As such difficulty increases it also becomes more difficult to separate the peptide of interest from unreacted substances and from side-products. Particularly the serine residue at position 4 of LH-RH and its derivatives tends to be racemized thus remains as impurities and so recovery of raw materials is impossible. Therefore the method is not technically satisfactory since posttreatment of the reaction is difficult and uneconomical.
The object of the invention is to provide a method for efficiently producing LH-RH derivatives in a large scale at lower costs.
As a result of our intensive researches to solve the abovementioned problems, we have achieved the present invention by finding a method for producing LH-RH derivatives suitable for industrial production by means of an enzymatic synthesis.
The present invention is characterized in that a peptide fragment shown by general formula (1):
pGlu-His-Trp-OR1xe2x80x83xe2x80x83(1)
(where R1 denotes lower alkyl) and a peptide fragment shown by general formula (2):
H-Ser-Tyr-X-Leu-Arg-Pro-Y (SEQ ID NO:4)xe2x80x83xe2x80x83(2)
(where X denotes an amino acid selected from the group consisting of D-Leu, D-Ser(But), D-Trp, (2-naphthyl)-D-Ala, and Gly; Y denotes Gly-NH2, Azgly (Azaglycine)-NH2 or NHR2 (where R2 is lower alkyl)) are allowed to react in the presence of an enzyme selected from the group consisting of chymotrypsin or chymotrypsin-like enzymes to produce a LH-RH derivative shown by general formula (3):
pGlu-His-Trp-Ser-Tyr-X-Leu-Arg-Pro-Y (SEQ ID NO:1)xe2x80x83xe2x80x83(3)
(where X and Y denote the same as in the above formula).
Here the present invention is characterized in that the abovementioned R1 is an alkyl group having 1 to 3 carbons and R2 is an alkyl group having 1 to 3 carbons. Further the present invention is characterized in that the enzyme selected from the group consisting of the chymotrypsin or chymotrypsin-like enzymes is chymotrypsin.
Moreover, the present invention is characterized in that a peptide fragment shown by general formula (4):
pGlu-His-Trp-OR1 xe2x80x83xe2x80x83(4)
(where R1 denotes lower alkyl) and a peptide fragment shown by general formula (5):
H-Ser-Tyr-X-Leu-Arg-Pro-Y (SEQ ID NO:4)xe2x80x83xe2x80x83(5)
(where X denotes an amino acid selected from the group consisting of D-Leu, D-Ser(But), D-Trp, (2-naphthyl)-D-Ala, and Gly; Y denotes Gly-NH2, Azgly-NH2 or NHR2 (where R2 is lower alkyl)) are allowed to react in the presence of an enzyme selected from the group consisting of chymotrypsin or chymotrypsin-like enzymes and in a solvent in which water or buffer is mixed with organic solvent to produce LH-RH derivatives shown by general formula (6):
pGlu-His-Trp-Ser-Tyr-X-Leu-Arg-Pro-Y (SEQ ID NO:1)xe2x80x83xe2x80x83(6)
(where X and Y denote the same as in the above formula).
Here the present invention is characterized in that the solvent wherein water or buffer and an organic solvent are mixed is a mixture in which water or buffer and an organic solvent miscible with water are mixed, or is a mixture in which water or buffer are saturated with an organic solvent partially miscible with water.
Moreover, the present invention is characterized in that a peptide fragment shown by general formula (7):
pGlu-His-Trp-OR1xe2x80x83xe2x80x83(7)
(where R1 denotes lower alkyl) and a peptide fragment shown by general formula (8):
H-Ser-Tyr-X-Leu-Arg-Pro-Y (SEQ ID NO:4)xe2x80x83xe2x80x83(8)
(where X denotes an amino acid selected from a group consisting of D-Leu, D-Ser(But), D-Trp, (2-naphthyl)-D-Ala, and Gly; Y denotes Gly-NH2, Azgly-NH2 or NHR2 (where R2 is lower alkyl)) are allowed to react in the presence of an immobilized enzyme that is selected from the group consisting of chymotrypsin or chymotrypsin-like enzymes and in a solvent in which water or buffer is mixed with an organic solvent to produce LH-RH derivatives shown by general formula (9):
xe2x80x83pGlu-His-Trp-Ser-Tyr-X-Leu-Arg-Pro-Y (SEQ ID NO:9)xe2x80x83xe2x80x83(9)
(where X and Y denote the same as described above).
In this specification, the LH-RH derivative is referred to as the one wherein Glycine at position 6 or at position 10 of the LH-RH shown by general formula (10) (SEQ. ID.NO:1):
PGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2xe2x80x83xe2x80x83(10)
is substituted by a different amino acid, a special amino acid or a modified amino acid. An amino acid at the position corresponding to Gly at position 6 (hereinafter referred to as X) may be a general D-amino acid or a modified D-amino acid. For example, the D-amino acid mentioned here may be D-Leu, D-rrp, D-Ala, D-Phe, D-Val, or D-His and the modified amino acid may be D-Ser(But) or (2-naphthyl)-D-Ala. In addition, X may be an L-amino acid. When X is a D-amino acid, it is preferably selected from the group consisting of D-Leu, D-Trp, D-Ala, D-Ser(But) and (2-naphthyl)-D-Ala and when X is an L-amino acid it is preferably Glycine. Glycine at position 10 (hereinafter referred to as Y) is preferably Gly-NH2, Azgly-NH2 or NHR2 (where R2 is lower alkyl).
xe2x80x9cLower alkylxe2x80x9d used herein means an alkyl having 1 to 3 carbons such as methyl, ethyl, propyl, or isopropyl. R1 is preferably a methyl or an ethyl group and R2 is preferably an ethyl or a methyl group.
Besides in this specification, abbreviations used for amino acids, peptides, protecting groups, solvents and others are according to rules of International Union of Pure and Applied Chemistry (IUPAC) and of International Union of Biochemistry (IUB) or to conventional symbols in the field the present invention pertains to. Examples are shown below. Possible optical isomers of amino acids indicate L- configuration unless otherwise indicated.
Tyar: Tyrosin residue
Gly: Glycine residue
Azgly: Azaglycine residue
Glu: Glutamic acid residue
pGlu: Pyroglutamic acid residue
Ser: Serine residue
Arg: Arginine residue
Pro: Proline residue
Leu: Leucine residue
His: Histidine residue
Ala: Alanine residue
Trp: Tryptophane residue
Et: Ethyl
Boc: t-Butoxycarbonyl
Aoc: t-Amyloxycarbonyl
Bz: Benzyl
Z: Benzyloxycarbonyl
Tos: Tosyl
OMe: Methyl ester
OBz: Benzyl ester
OSu: N-Hydroxysuccinimide ester
TFA: Trifluoroacetic acid
THF: Tetrahydrofuran
DMF: Dimethylformamide
DCC: Dicyclohexylcarbodiimide
WSC: N-ethyl-Nxe2x80x2-dimethylaminopropyl-carbodiimide
HOSu: N-hydroxysucciniimide
HOBt: 1-Hydroxybenzotriazol
MeOH: Methanol
EtOH: Ethanol
AcOH: Acetic acid
The peptide fragment shown by the general formula (1) corresponds to amino acid residues at positions 1 to 3 of the amino acid sequence of the LH-RH derivative shown by the general formula (3). Further, the peptide fragment shown by the general formula (2) corresponds to amino acid residues at positions 4 to 10 of the amino acid sequence of the LH-RH derivative shown by the general formula (3).
Each of the peptide fragments shown by the general formula (1) or shown by the general formula (2) can be synthesized according to a well-known method for synthesizing peptides. For example, according to the methods described in The Peptides. Vol 1, Schreder and Luhke., 1966., Academic Press, New York, U.S.A., or in Peptide Synthesis, Izumiya et.al., 1975, Maruzen Corporation, peptide fragments can be synthesized by the azido method, acid chloride method, acid anhydride method, mixed acid anhydride method, DCC method, activated ester methods (e.g., p-nitrophenyl ester method, N-hydroxysuccinimide ester method, and cyanomethyl ester method), method using Woodword""s Reagent K, carboimidazole method, oxidation-reduction method, DCC-additive (HONB, HOBt, HOSu) method, and solid phase method. With these general methods for synthesizing peptides, peptides can be produced for example by a so-called stepwise elongation method wherein one amino acid is in order condensed with a C-terminal amino acid according to the amino acid sequence or by the fragment condensation method wherein fragments each composed of several fragments are synthesized and coupled to each other.
In the reaction process for synthesizing the peptide fragments shown by the above general formula (1) or (2), functional groups that are not involved in the reaction are protected by normal protective groups, being eliminated after the completion of the reaction. Furthermore, functional groups involved in the reaction are normally activated. Each of these reaction methods is well known and the reagents used therein can be properly selected from well-known reagents.
The protective groups of amino groups include, for example, benzyloxycarbonyl (Z), t-butyloxycarbonyl (Boc), t-amyloxycarbonyl (Aoc), isobonyloxycarbonyl, p-methoxybenzyloxycarbonyl, 2-chloro-benzyloxycarbonyl, adamantyloxycarbonyl, trifluoroacetyl, phthaloyl, formyl, o-nitrophenylsulphenyl, and diphenylphosphinothioyl. Normally Boc is used to protect amino groups. But when D-Ser (But) is coupled, the use of Z group allows to remove only Z group without removing But (t-butyl) group.
The protective groups of carboxyl groups include, for example, alkylesters (for example, methyl ester, ethyl ester, propyl ester, butyl ester, and tert-butyl ester), benzyl ester, p-nitrobenzyl ester, methyl benzyl ester, p-chlorobenzyl ester, benzhydryl ester, benzyloxycarbonyl hydrazide, tert-butyloxycarbonylhydrazide, and tritylhydrazide. To hydrazidize, methyl ester or ethyl ester is preferably used.
Activated carboxyl groups involved in peptide synthesis include, for example, acid chloride, acid anhydride, mixed acid anhydride, azide, activated esters (for example, esters of pentachlorophenol, p-nitrophenol, N-hydroxysucciniimide, N-hydroxybenztriazole, and N-hydroxy-5-norbornene-2,3-dicarboxyimide). Among them the azide method with less racemization tendency is preferably used upon condensation of fragments.
In addition, peptide bond synthesis reaction may be performed in the presence of condensing agents, such as carbodiimide reagents including dicyclohexyl carbodiimide, and carbodiimidazole or in the presence of tetraethylpyrophosphate.
Usually proteolytic enzymes have been mainly used to cleave peptide bonds. Further it has been well known from old times that proteolytic enzymes can also be involved in the reverse reaction to synthesize peptide bonds. Synthesis of long-chain peptides with enzymes is limited to a case that the structure contains amino acids having a limited substrate specificity or in a case that an enzyme has a limited substrate specificity. Thus general enzymes, such as trypsin and chymotrypsin, are rarely used in peptide synthesis reactions. Accordingly, enzyme reaction is mainly used for relatively short-chain peptide bonds, such as oligopeptides, but it still takes time for examining synthesis conditions. Because of this, chemical synthesis is generally used to produce peptides, and enzymatic synthesis is tried only when in chemical synthesis many side reactions occur or reactions are difficult. Enzymatic synthesis can be extremely useful, when the synthesis is performed under mild conditions that enables mass production and the above-mentioned problems in chemical synthesis are solved with appropriate conditions.
As a result of intensive researches, the present inventors have succeeded in effectively producing LH-RH derivatives shown by the general formula (3) by allowing the peptide fragment shown by the general formula (1) and the one shown by the general formula (2) to react in the presence of chymotrypsin or a chymotrypsin-like enzyme so as to couple to each other.
Chymotrypsin utilized in the present invention is a kind of serine protease that is registered as enzyme number EC.3.4.21.1 of International Union of Biochemistry (IUB), Enzyme Committee, and is obtained from bovine pancreas. Chymotrypsin is on market and sold by SIGMA Corporation, etc.
Chymotrypsin-like enzymes are proteolytic enzymes that recognize aromatic amino acids such as Tyr and Phe, and for example include xcex1-chymotrypsin.
Chymotrypsin or chymotrypsin-like enzymes are used preferably because one of the recognition sites of chymotrypsin or chymotrypsin-like enzymes, Trp, is present in LH-RH derivatives.
Reaction of chymotrypsin or chymotrypsin-like enzymes are performed normally in a medium containing a buffer with a pH of about 5 to about 10, preferably pH of about 6 to about 9, more preferably pH of about 7.5 to about 8.5.
The term xe2x80x9cmedium containing a bufferxe2x80x9d used herein refers to a solvent having a buffer itself as a medium, a mixture of a buffer (selected from the various ones explained next) and an organic solvent miscible with water, or a mixture of such a buffer and an organic solvent partially miscible with water.
The buffer is not particularly limited and various buffers can be used as far as the pH is within the above range. For example, the buffer includes Tris-hydrochloric acid, MacIlvaine""s buffer, phosphate buffer, ammonium acetate buffer, Atkins and Pantin buffer and Veronal buffer.
When the buffer is used as a reaction medium, the buffer is usually used by mixing with an organic solvent miscible with water. The organic solvents miscible with water include dimethylformamide (DMF), dimethylsulfoxide (DMSO), dimethylimidazolidinone (DMI), hexamethylphosphoryltriamide (HMPA), methanol (MeOH), ethanol (EtOH). Dimethylformamide, methanol, and ethanol are preferable.
In addition, an organic solvent partially miscible with water such as n-butanol (1-BuOH) or ethyl acetate (EtOAc) can be used. In the use of such an organic solvent, BuOH is preferably used to facilitate the operation of a partition chromatography in the posttreatment.
These organic solvents can be used alone or in combination of the two or more of them.
When the reaction of chymotrypsin and chymotrypsin-like enzymes is performed in a mixture of water or buffer and organic solvent, not in a solvent consisted only of water or buffer, the yield of a peptide of interest can significantly be improved.
Generally, the mixing ratio of the organic solvent miscible with water to water or buffer is preferably 50 volume % or less in view of the reactivity. When an immobilized enzyme, such as chymotrypsin immobilized with celite, is used, the preferable mixing ratio is 80 volume % or more. When an organic solvent partially miscible with water is used, there is an advantage that the use of the organic solvent saturated with water results in high reaction efficiency of enzymes.
The above described reaction of enzymes is performed within a temperature range wherein chymotrypsin or chymotrypsin-like enzymes can act, normally about 0xc2x0 C. to about 50xc2x0 C., preferably about 0xc2x0 C. to about 20xc2x0 C. The reaction at about 10xc2x0 C. has an effect capable of inhibiting a hydrolysis reaction that occurs simultaneously.
Amount of chymotrypsin or of chymotrypsin-like enzymes to be used is not specifically limited and can be varied depending on reaction conditions. For example, the use of about 50 mg to about 100 mg of chymotrypsin based on 50 g of a substrate leads to the synthesis of a target peptide in a yield ranging from about 75 to about 85% after 1-hour reaction.
Normally 1 to 5 moles, or preferably 2 to 4 moles, of the peptide fragment shown by general formula (1) is used per mole of the peptide fragment shown by general formula (2).
In the method of the present invention, chymotrypsin or chymotrypsin-like enzymes dissolved in water or an appropriate buffer can be used, or those immobilized by general methods including the carrier-coupling method, crosslinking method, entrapping method, and other methods can be used as immobilized enzymes. The carriers used in the carrier-coupling method include polysaccharide derivatives, such as cellulose, dextrun, and agarose; polyacrylamide gel; celite; and porous glass. The reagents for crosslinking in the crosslinking method, for example, include glutaraldehyde, bisdiazobenzidine, N,N-polymethylenebisiodoacetamide, and N,N-ethylene bismaleinimide. The materials used in the entrapping method include, for example, polyacrylamide gel, polyacrylalcohol gel, starch, konnyaku powder, nylon, polyurea, polystyrene, ethyl cellulose, colodion, and cellulose nitrate. However, the immobilization method is not limited to those using the above methods.
In the production method according to the present invention, LH-RH derivatives shown by the following formula (11) can be obtained for example in the manner described below:
pGlu-His-Trp-Ser-Tyr-D-Ser(But)-Leu-Arg-Pro-NHEt (SEQ ID NO:1)xe2x80x83xe2x80x83(11)
That is, a given amount of a peptide fragment shown by the following formula (12)
pGlu-His-Trp-OMexe2x80x83xe2x80x83(12)
and a given amount of a peptide fragment shown by the following formula (13)
H-Ser-Tyr-D-Ser(But)-Leu-Arg-Pro-NHEt (amino acids 4-11 of SEQ ID NO:1)xe2x80x83xe2x80x83(13)
are stirred in n-butanol saturated water at a pH of around 7.8 at about 10xc2x0 C. for 1 hour in the presence of chymotrypsin having a given activity, to obtain the LH-RH derivative. Preferable activity of chymotrypsin is about 1000 or more international unit (U)/mg in order to facilitate the removal thereof in the posttreatment. A high yield can be obtained in the reaction using the molar ratio 3:1 of the peptide fragment shown by formula (12) to the one shown by (13). In addition, as an alternative for the addition of enzymes dissolved in water or buffer to the reaction fluid, for example, chymotrypsin immobilized with agarose according to conventional methods can be used as an immobilized enzyme. The use of immobilized enzymes facilitates removal of them by filtering the reaction fluid through a filter such as a glass filter after the completion of the reaction and allows a reuse of them.
The LH-RH derivatives can be obtained as described above and the obtained LH-RH derivatives can be purified as follows.
The LH-RH derivatives produced by the method according to the present invention can be desalted and purified by general methods. Desalting can be performed by various methods, which utilize difference in molecular sizes between the derivatives and the salt, including gel filtration, ultrafiltration, and dialysis. For example, it can be performed by ultrafiltration using a membrane made of cellulose acetate, gel filtration using Sephadex column such as Sephadex LH-20, Sephadex-60, Sephadex G-25, and ion-exchange chromatography using e.g., DEAE-cellulose. In purification, liquid chromatography including partition chromatography wherein two types of liquids that are not miscible with each other (e.g., water and n-butanol) are employed and the difference in partition coefficients between the two liquid phases is utilized, normal phase chromatography using a solid phase such as silica gel, and reversed phase chromatography using a solid phase such as ODS-silica gel can be used by such as high performance liquid chromatography (HPLC).
For example, since the LH-RH derivative produced by the methods according to the present invention is hydrophilic, the derivatives can be purified by using organic solvent that is not completely miscible with water as shown below. The mixture reacted as described above is extracted by using the organic solvent, partitioned by adding water to the extracted fluid, then solidified with a solvent such as ether. A preferable organic solvent for the extraction is n-butanol (n-BuOH). Such a solvent can lead high extractability because of its high polarity and it is not miscible with water, so that it is convenient for the subsequent partition chromatography process.
Three hundred ml of n-butanol (about xc2xc of the volume of the reaction mixture) is added to the reaction mixture (1200 ml) to perform partition chromatography between the n-butanol and the water in the reaction mixture. Extraction with n-butanol can be repeated if necessary. The aqueous layer and the organic layer are concentrated separately. The above peptide fragment (7) recovered from the aqueous layer can be reused for the later reaction.
It is preferable that the roughly purified product obtained from the organic layer by partition chromatography solidifies by using a solvent such as diethyl ether or ethyl acetate, because LH-RH derivatives are slightly soluble in such a solvent. Then the solidified product is dissolved in an appropriate buffer like ammonium acetate, and fractioned by column chromatography with a proper solid phase and a mobile phase. The use of a weak cation exchange resin such as CM cellulose or a strong cation resin such as SP allows to obtain interaction with the Arg residues contained in LH-RH. The use of a buffer (which is the same buffer used when the solidified roughly purified product is dissolved) as a mobile phase has an advantage of resulting in a high yield when the salt concentration is increased in a linear gradient. For example, 0.01M aqueous ammonium acetate solution and 0.1M aqueous ammonium acetate solution can be used. The resulting eluate can be purified with HPLC properly selecting a solid phase and an eluent. In HPLC, ODS silica column, e.g. TSK gel ODS-120T, can be used as a solid phase and the use of ODS silica column is the most suitable. Furthermore, 0.1% TFA-acetonitrile can be used as a mobile phase. Here, the content of acetonitrile is increased by 1% per minute from the beginning 0.1% TFA-20% acetonitrile so as to obtain a linear gradient of 0.1%TFA-50%acetonitrile, then the target LH-RH derivative can be separated well. Purification is not limited to the abovementioned methods. Moreover, when immobilized enzymes are used, ion exchange can be performed without subjecting to partition chromatography.
The method for producing LH-RH derivatives according to the present invention is more useful industrially because it has advantages as follows compared with known conventional methods.
i) Since LH-RH derivatives can be synthesized without side reactions (e.g., racemization) because of the property of an enzymatic reaction, the LH-RH derivatives can be easily purified and separated.
ii) A high yield of an LH-RH derivative can be obtained. Moreover unreacted fragments can be recovered and reused thus the method is economically advantageous.
The LH-RH derivatives may be changed to pharmaceutically acceptable salts, such as their acetate, hydrochloride and phosphate, if necessary.